One of the greatest challenges facing the development of improved cancer therapeutics is the need to selectively kill all of the cancer cells in a patient without harming normal cells. To achieve this high level of selectivity, the genetic concept of synthetic lethality offers a promising strategy. This concept is based on the observation that mutations in two different genes that both contribute an essential biochemical pathway, such as the genes BRCA1/2 and PARP, can be exploited to make certain cancers uniquely sensitive to anticancer agents. In this mechanism, disruption of either gene alone does not affect cellular viability, but agents or mutations that affect both genes are lethal. Based on our preliminary research results, we propose here to extend the concept of synthetic lethality to the targeting of pairs of growth factor receptors that drive the proliferation of highly aggressive cancers. To accomplish this objective, we will create novel antibody conjugates, similar in structure to the FDA-approved anticancer antibodies Kadcyla and Adcetris, designed with the unique ability to synergistically kill cancer cells that express two distinct cell surface receptors. To enable this cytotoxic synergy, one targeting antibody will be linked via a disulfide to a cell-impermeable cytotoxin that is incapable of unaided passage across cellular membranes. When this first antibody binds a specific growth factor receptor on the cell surface, and is internalized by endocytosis, the stability of the disulfide, in conjunction with the cell-impermeability of the cytotoxin, will cause entrapment in membrane- sealed endosomes. This entrapment will prevent toxicity unless membranes of these endosomes are disrupted by co-administration with a secondary agent. To synergistically kill cancer cells, this antibody conjugate will be co-administered with a second anti-growth factor antibody linked to a non-toxic endosome disruptive peptide. Release of this peptide from the second antibody will form pores in endosomal membranes. These pores will enable cytosolic glutathione to enter endosomes, break the disulfide bond linking the toxin to the first antibody, and activate toxicity by enabling escape of the cytotoxin into the cytoplasm. This unique design of antibody conjugates will provide high selectivity for killing specific cancer cells that express two distinct cell surface receptors without affecting normal cells that express one of these two target proteins. This novel approach, termed here synthetic lethal targeting, will be pursued by the synthesis of masked cytotoxins and endosome disruptive peptides, the elucidation of mechanisms of pore formation in endosomal membranes, conjugation of these agents to antibodies that bind the growth factor receptors EGFR, HER2, and HER3, and evaluation of efficacy in vitro and in mouse models of cancer. Given that co-expression of HER2/EGFR and HER2/HER3 in breast cancer, and EGFR/HER3 in lung and pancreatic cancer, drives the proliferation of some of the most incurable cancers, extending synthetic lethality by targeting two distinct growth factor receptors could provide a less toxic platform to eradicate cancer across cell types.